There are many known lipophilic bioactive substances useful for living organisms. Among those, coenzyme Q is an essential component widely distributed in living organisms from bacterium to mammal, and is known as a component constituting the electron transport system of mitochondria in the cell of living organisms. Coenzyme Q functions as a transport component in the electron transport system by repeating oxidation and reduction in mitochondria and, of coenzyme Q, reduced coenzyme Q is known to have an antioxidant action. In human, coenzyme Q10, which is a coenzyme Q having 10 repeat structures in the side chain, is the main component, and generally, about 40-90% thereof in living organisms is of the reduced type. The physiological actions of coenzyme Q include activation of energy production by activating mitochondria, activation of cardiac function, cellular membrane stabilizing effect, cell protection effect by antioxidant action and the like.
Of coenzyme Q10, oxidized coenzyme Q10 has conventionally been used as a drug for congestive heart failure or health food and, in recent years, reduced coenzyme Q10 having a higher physiological activity is known.
Lipophilic bioactive substances such as coenzyme Q10 and the like can be obtained, for example, by synthesis, fermentation, extraction from naturally occurring substances and the like. Where necessary, coenzyme Q10 having a higher purity can also be obtained by subjecting the obtained extract to purification by chromatography, or crystallization by crystal precipitation. For example, a general method of obtaining coenzyme Q10 includes culturing a coenzyme Q10-producing microorganism, and extracting coenzyme Q10 in the microorganism from a suspension of the microorganism by using an organic solvent.
For an operation to extract a useful component contained in a microbial cell, a method including dehydrating an aqueous suspension of a cultured microorganism to give a wet fungus body and bringing same into contact with an organic solvent, a method including dehydrating an aqueous suspension, drying same to give a dry fungus body and bringing same into contact with an organic solvent, and a method including bringing an aqueous suspension directly into contact with an organic solvent, and performing a liquid-liquid extraction are conventionally known.
Patent document 1 describes an example wherein a suspension of cultured Phaffia yeast was centrifuged to recover fungus, recovered fungus body was spray dried, and astaxanthin in the fungus body was extracted while homogenizing with a mixed solvent of hexane, ethanol and the like. In addition, an example wherein a fungus body of the genus Mortierella was cultured, a suspension of the fungus body was dehydrated and dried and an arachidonic acid-containing oil was extracted with hexane (patent document 2), and an example wherein a fungus body of the genus Mucor was cultured, the culture medium was homogenized, freeze-dried and γ-linolenic acid was extracted with a solvent such as hexane and the like (patent document 3) are known. In these extraction methods, a dry fungus body and an organic solvent as an extraction solvent are mixed, and a solid-liquid separation is performed after completion of the extraction operation to remove fungus body residues, whereby an organic phase containing the object substance can be obtained.
Patent document 4 discloses an example wherein a wet fungus body or dry fungus body of a coenzyme Q10-containing a microorganism was brought into contact with methanol at a low temperature, contaminants inside and outside the fungus body were removed, and then the fungus body was brought into contact with methanol at a high temperature, whereby coenzyme Q10 was extracted. Such solid-liquid extraction operation is advantageous since a solid-liquid separation after extraction is easy due to a large difference in the specific gravity between a fungus body and an extraction solvent, the loss of the object substance is small, and extraction can be performed with high efficiency.
On the contrary, these methods have problems in that they require a step of dehydrating and drying, before extraction, a large amount of water from an aqueous suspension of a cultured microorganism by an apparatus such as centrifugation, spray drier, freeze drier and the like, a sufficient extraction rate may not be achieved in some cases depending on the content of water remaining in the fungus body, the apparatus cost and operating cost become high and the like.
As an example wherein an aqueous suspension of a microorganism is extracted without dehydration and drying, an example wherein a homogenized suspension of a cultured microorganism was contacted with an organic solvent such as hexane, 2-propanol and the like, whereby coenzyme Q10 in the fungal body was extracted, is known (patent document 5). In such a liquid-liquid extraction operation, an object substance can be extracted in a high yield and in a large treatment amount without dehydrating and drying the microorganism. However, when an aqueous suspension of a microbial cell or a cell homogenate thereof, particularly an aqueous suspension of the cell homogenate, especially an aqueous suspension of the cell homogenate by a physical treatment, is subjected to extraction with an organic solvent, problems occur since an emulsion phenomenon and the like tend to occur due to the presence of cell components such as partial protein and the like, a large amount of an organic phase containing the object substance is trapped in a homogenate suspension of the microbial cell, the organic phase cannot be separated efficiently, which in turn not only decreases the yield but also lowers the recovery rate of the extraction solvent used. Moreover, complicated operation and increase of costs have been the problem, since, besides the hydrophobic organic solvent, a hydrophilic organic solvent needs to be used in an amount exceeding a certain level.
Conventionally, when a surfactant is used for liquid-liquid extraction, the affinity between an aqueous phase and an organic solvent phase becomes high due to an interface effect, as a result of which homogenizing easily occurs, and the aqueous phase and the organic solvent phase require a long time for separation, or often do not separate even when time is spent thereon, and in some cases, require a forcible separation by an operation such as centrifugation and the like. Therefore, use of a surfactant in an extraction step in general liquid-liquid extraction has been considered to be unpreferable.